We have continued our studies on the biochemical properties of the motility of malignant tumor cells in as much as migration is a requirement for their metastasis. The preparation of an autocrine motility factor (AMF) from A2058 human melanoma cells has been scaled up using successive separation procedures with hydrophobic interaction and HPLC anion exchange chromatography. Studies on the mechanism of action of AMF have indicated that it may stimulate motility by activating the phosphatidyl inositol (PI) pathway. AMF enhanced both migration and PI turnover in a parallel, concentration-dependent manner. AMF also stimulated release of arachidonic acid in melanoma cells. The peptide mellitin increased adherence of cells as well as stimulating phospholipase A2 activity. AMF was also found to increase Ca++ fluxes in cells at early times. These results collectively implicate Ca++ mobilization and lipid metabolic pathways as signal processing components of the motile response in tumor cells. We have also demonstrated that insulin-like growth factors stimulate migration in melanoma cells and accomplish this via a pertussis-toxin insensitive pathway. This is in contrast to the mechanism of action of AMF which is toxin-sensitive. The significance of this may be that malignant tumor cells have alternate ways of mounting a motile response to diverse chemical stimuli, a property which could facilitate their metastatic dissemination in the host. Cell lines other than melanoma, such as ovarian carcinoma, breast and bladder cancer, and a rodent line, were shown to produce AMFs. Some of these cell types and A2058 melanoma cells respond to each other's AMF, suggesting that there may be homologous active site domains within AMFs in general. The definitive determination of this point must await sequencing AMF, a result most likely to come when the AMF gene is cloned.